International Journal of

Molecular Sciences Article

A Novel Therapeutic Approach in the Treatment of Pulmonary Arterial Hypertension: Allium ursinum Liophylisate Alleviates Symptoms Comparably to Sildenafil Mariann Bombicz 1 , Daniel Priksz 1 , Balazs Varga 1 , Andrea Kurucz 1 , Attila Kertész 2 , Akos Takacs 1 , Aniko Posa 3 , Rita Kiss 1 , Zoltan Szilvassy 1 and Bela Juhasz 1, * 1

2 3

*

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; [email protected] (M.B.); [email protected] (D.P.); [email protected] (B.V.); [email protected] (A.K.); [email protected] (A.T.); [email protected] (R.K.); [email protected] (Z.S.) Department of Cardiology, Faculty of Medicine, University of Debrecen, H-4032 Debrecen, Hungary; [email protected] Department of Physiology, Anatomy and Neuroscience, Faculty of Science and Informatics, University of Szeged, Kozep Fasor 52, H-6726 Szeged, Hungary; [email protected] Correspondence: [email protected]; Tel.: +36-5242-7899 (ext. 56109)

Received: 9 June 2017; Accepted: 27 June 2017; Published: 4 July 2017

Abstract: Right-sided heart failure—often caused by elevated pulmonary arterial pressure— is a chronic and progressive condition with particularly high mortality rates. Recent studies and our current findings suggest that components of Wild garlic (Allium ursinum, AU) may play a role in reducing blood pressure, inhibiting angiotensin-converting enzyme (ACE), as well as improving right ventricle function in rabbit models with heart failure. We hypothesize that AU may mitigate cardiovascular damage caused by pulmonary arterial hypertension (PAH) and has value in the supplementary treatment of the complications of the disease. In this present investigation, PAH was induced by a single dose of monocrotaline (MCT) injection in Sprague-Dawley rats, and animals were divided into 4 treatment groups as follows: I. healthy control animals (Control group); II. pulmonary hypertensive rats (PAH group); III. pulmonary hypertensive rats + daily sildenafil treatment (Sildenafil group); and IV. pulmonary hypertensive rats + Wild garlic liophylisate-enriched chow (WGLL group), for 8 weeks. Echocardiographic measurements were obtained on the 0 and 8 weeks with fundamental and Doppler imaging. Isolated working heart method was used to determinate cardiac functions ex vivo after thoracotomy on the 8th week. Histological analyses were carried out on excised lung samples, and Western blot technique was used to determine Phosphodiesterase type 5 enzyme (PDE5) expression in both myocardial and pulmonary tissues. Our data demonstrate that right ventricle function measured by echocardiography was deteriorated in PAH animals compared to controls, which was counteracted by AU treatment. Isolated working heart measurements showed elevated aortic flow in WGLL group compared to PAH animals. Histological analysis revealed dramatic increase in medial wall thickness of pulmonary arteries harvested from PAH animals, but arteries of animals in sildenafil- and WGLL-treated groups showed physiological status. Our results suggest that bioactive compounds in Allium ursinum could have beneficial effects in pulmonary hypertension. Keywords: Allium ursinum; pulmonary arterial hypertension; phosphodiesterase; sildenafil; monocrotaline

Int. J. Mol. Sci. 2017, 18, 1436; doi:10.3390/ijms18071436

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1. Introduction Right-sided heart failure—often caused by pulmonary hypertension resulting from elevated arterial pressure—is a chronic and progressive condition with particularly high mortality rates. Pulmonary arterial hypertension (PAH) is a vascular dysfunction characterized by abnormalities of endothelial and smooth muscle cells of pulmonary vessels. As a result of the processes, vascular resistance increases, which quickly starts depleting compensatory mechanisms in the right ventricle. This maladaptation eventually leads to irreversible damage of the cardiovascular system and premature death of patients suffering from the disease. PAH itself essentially represents a vasculopathy, and a chronic, progressive disease entity. This is reflected in the current etiology and pathophysiology-based clinical classification by the World Health Organization (WHO) of the types of pulmonary hypertension (PH), where PAH represents one individual group [1]. The condition is predominantly characterized by progressively-increasing vascular resistance (PVR) of pulmonary arteries. Since the right ventricle is highly sensitive to changes in afterload, adaptive mechanisms to increased pressure—such as Frank-Starling mechanism and ventricular muscle hypertrophy—appear in early stages of the disease [2]. Increased tension in chordae tendinae and dilatation of the annulus leads to tricuspid regurgitation, resulting in dilation of the right atrium, right ventricular volume overload and decreased cardiac output [3]. Ischemic lesions also appear due to worsening hypethrophy. The interventricular septum becomes thinner and is even pressed into the left side of the heart, thereby reducing the volume of the left ventricle during diastole. The processes necessarily result in decreased cardiac output caused by abnormal reduction of the left ventricular preload. This vicious circle ends in systemic hypoxia, severe heart failure and premature death [4]. The first line of underlying pathological processes in PAH is vasoconstriction of pulmonary arteries caused by an imbalance of vasoconstrictor and vasodilator mediators. Multiple dysregulated signalling pathways have been identified in PAH patients, and a significant portion of disease-specific therapeutic agents have an influence on the course of the disease through these routes, namely the nitric oxide (NO) pathway (I), the prostacyclin (II) and endothelin-1 pathways (III) [5–8]. Evidences include decreased NO synthase expression and abnormally high arginase enzyme levels in lungs of PAH patients, as well as overexpression of phosphodiesterase type 5 enzyme (PDE5), therefore reduced cyclic guanosine monophosphate (cGMP) levels [9,10]. Further studies have shown decreased levels of prostacyclin (PGI2) and cyclic adenosine monophosphate (cAMP) in the relevant tissues, which is partly explained by the fact that the expression of prostacyclin synthase is abnormally low in the lungs of these patients [11,12]. Endothelin-1, acting on ETA and ETB receptors, is a vasoactive mediator with concominant proliferative effects on smooth muscle cells in the vessel wall. Under physiological conditions, when endothelium is intact, activation of ETB receptors increases the production of NO and PGI2. However, in PAH patients, the ETA receptor pathway overwhelms these effects, causing vasoconstriction by increasing intracellular calcium levels and activation of protein kinase C enzyme (PKC), as well as inducing proliferation via the MAPK (mitogen-activated protein kinase) pathway in smooth muscle cells of pulmonary vessels. Elevated endothelin-1 levels were identified in affected pulmonary tissues, and the peptide is presented in higher concentrations in PAH patients compared to healthy controls. Moreover, strong correlation has been found in the levels of endothelin-1 and the degree of pulmonary hypertension [13,14]. The first modern therapeutic approach based on the previous facts, was to elevate cGMP levels in the lung, since the required vasodilation caused by NO is mediated by cGMP as a secondary messenger. Since therapeutic admininstration of nitric oxide itself by inhalation is possible but very inconvenient, elevation of cGMP level is more feasible by blocking its metabolization through the inhibition of PDE enzymes. Phosphodiesterase type 5 is the dominant enzyme isoform in the lung that converts cGMP into guanosine monophosphate (GMP), and thus blocking this isoenzyme prevents cGMP breakdown and elevates its intracellular levels [15]. PDE5 has also been found to be overexpressed in pulmonary tissue samples of PAH patients. PDE5 inhibitors (PDE5I) increase the activity of NO-cGMP pathways,

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and thereby promote vasodilation and even have antiproliferative effects on smooth muscle cells of pulmonary vessels. Sildenafil-citrate, the former blockbuster PDE5I for therapy of erectile dysfunction, was approved by the FDA (U.S. Food and Drug Administration) to treat pulmonary hypertension in the year 2005 [16,17]. Sildenafil has clear benefits in PAH, however, the FDA warns about its long-term use in children because of elevated risk of mortality as revealed in the STARTS-2 trial [18]. Wild garlic (Allium ursinum L.), a plant distributed widely in Eurasia and also known as bear’s garlic or buckrams, is a popular dietary component, spice and also an element of traditional medicine. Unfortunately, wild garlic, due to the similarity of the leaves, can easily be mistaken with Colchicum autumnale, a toxic, colchicine-containing plant, thus some case-studies report rare, but serious poisonings [19]. The herb has a garlic-like scent due to sulfur-containing compounds and also contains high levels of polyphenol derivatives, mostly flavonoid glycosides [20]. The majority of pharmacological properties of the plant extract or liophylisate are comparable to those of the close relative, cultivated garlic (Allium sativum), however, some effects of wild garlic are superior or unique, possibly due to the presence of some specific components, such as phytosterols and a galactolipid-derivative (1,2-di-O-α-linolenoyl-3-O-β-D-galactopyranosyl-sn-glycerol). This component seems to be specific for Allium ursinum and may be absent in other Allium species [21]. While a large amount of data is available on the characteristics of A. sativum, possible therapeutic effects of A. ursinum are poorly studied, and only a few publications have investigated pharmacological properties of this plant. Antiaggregatory effects on human platelets are well-proven [21,22], as well as significant in vitro inhibition of 5-lipoxygenase (5-LOX) and cyclooxygenase (COX) enzymes [23]. Bioactive components of wild garlic have fairly high antioxidant and free radical scavenger activity, mainly due to superoxide-dismutase, catalase and peroxidase activity of the bulb and leaves [24]. Wild garlic extracts containing ajoene, methyl-ajoene, allicin, diallyl-disulfide have also shown marked inhibition on cholesterol biosynthesis in vitro, equivalently to cultivated garlic [25]. Allium ursinum preparations reduce blood pressure and inhibit angiotensin-converting enzyme (ACE) in vivo when tested on spontaneously hypertensive (SHR) rats [26,27]. Capability of ACE inhibition has been further evidenced by other studies, where angiotensin-converting-enzyme inhibitor (ACEI) activity of wild garlic was even found to be superior than such effect of regular garlic [25]. Antihypertensive, cholesterol-lowering, and ACEI effects contribute to cardioprotection, and such property of the plant was investigated in a rat model of ischemia/reperfusion injury. Hearts of rats treated with wild garlic extract showed reduced incidence of ventricular fibrillation and tachycardia with decreased ischemic areas in myocardial tissues [28]. In a rabbit model of hypercholesterolemia-induced heart failure, our research team previously demonstrated that supplementation with Wild garlic improves right ventricle systolic function measured by tricuspid annular plane systolic excursion (TAPSE) [29]. Moreover, studies suggest that bioactive saponines and flavonoids of other Allium species (A. chinese, A. cepa) have phosphodiesterase enzyme (PDE 5A) inhibitor properties [30,31]. According to the aforementioned findings, we hypothesize that bioactive components of Allium ursinum may have benefit in the treatment of pulmonary hypertension, due to possible effects on phosphodiesterase enzyme system in myocardial and pulmonary tissues. 2. Results 2.1. Mass Spectrometry The quasi molecular ions cationized by potassium and sodium were observed. It can be assumed that, based on the the mass of [M + K]+ or [M + Na]+ peaks, the following compounds may be present in the plant, as seen in Table 1: kaempferol-3-O-rutinoside (at m/z 617.4 [M + Na]+ and m/z 633.3 [M + K]+ ); quercitrin (at m/z 471.2 [M + Na]+ and m/z 487.2 [M + K]+ ); juglanin at m/z 441.4 [M + Na]+ and m/z 457.3 [M + K]+ ; dracorubin (at m/z 511.2 [M + Na]+ and m/z 527.2 [M + K]+ ); and blumeatin (at m/z 325.2 [M + Na]+ , m/z 341.2 [M + K]+ and m/z 303.1 [M + H]+ ).

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Table 1. Mass spectrometric analysis of the Wild garlic (Allium ursinum) samples. Table 1 shows measured and exact m/z values of the main compounds present in A. ursinum leaf liophylisate. Structure

PubChem CID

74640 74640 46189 46189 47726 47726 68247 68247 48987 48987

24211973 24211973 5280459 5280459 5748554 5748554 160270 160270 11289628 11289628

Common Name Kaempferol-3-O-rutinoside Kaempferol-3-O-rutinoside Quercitrin Quercitrin Juglanin Juglanin Dracorubin Dracorubin Blumeatin Blumeatin

Input m/z

Exact m/z

∆

Formula

Ion

617.4 633.3 471.2 487.2 441.4 457.3 527.2 511.2 341.2 325.2

617.1472 633.1212 471.0898 487.0637 441.0792 457.0531 527.1255 511.1516 341.0422 325.0683

0.2528 0.1788 0.1102 0.1363 0.3208 0.2469 0.0745 0.0484 0.1578 0.1317

C27 H30 O15 Na C27 H30 O15 K C21 H20 O11 Na C21 H20 O11 K C20 H18 O10 Na C20 H18 O10 K C32 H24 O5 K C32 H24 O5 Na C16 H14 O6 K C16 H14 O6 Na

[M + Na]+ [M + K]+ [M + Na]+ [M + K]+ [M + Na]+ [M + K]+ [M + K]+ [M + Na]+ [M + K]+ [M + Na]+

Calculations were made on the basis of molar mass of the K and Na adducts. The exact molar mass of each compound was calculated as follows: measured m/z minus the molar mass of the adducted ion equals the molar mass of the compound. 2.2. Echocardiography All echocardiographic examinations were completed within a 20-min time interval, and all animals managed to recover from the anesthesia with stable heart rates and respiratory frequencies during the whole procedure. As seen in Table 2, Fractional Shortening (FS) and Ejection Fraction (EF) values did not show any changes among groups. Systolic function estimated by measuring mitral annular plane systolic excursions (MAPSE) showed no changes among treatment groups, a result also observed for Heart rate (HR). Table 2. Echocardiographic data of the four rat test groups in the study. Right ventricle function was estimated using tricuspid annular plane systolic excursion (TAPSE). Significant decreases were found in TAPSE values of PAH (pulmonary arterial hypertension) group animals compared to the Controls. Values for animals receiving sildenafil injection and Wild garlic (Allium ursinum) liophylisate-enriched chow (WGLL) were elevated in comparison to the PAH group. Parameter

Control

PAH

Sildenafil

WGLL

LV Ejection Fraction (%) LV Fractional Shortening (%) LV mass (g) Stroke volume (mL) HR (bpm) LVOT maxPG (mmHg) LVOT meanPG (mmHg) LVOT Vmax (m/s) LVOT Vmean (m/s) Lat S’ (cm/s) MV E vel (cm/s) MV A vel (cm/s) MV E/A ratio MAPSE (mm) TAPSE (mm)

73.39 ± 3.638 38.64 ± 3.268 1.388 ± 0.085 0.459 ± 0.070 269.5 ± 16.21 2.225 ± 0.247 1.032 ± 0.112 0.753 ± 0.041 0.432 ± 0.025 32.29 ± 1.782 65.81 ± 3.860 40.15 ± 4.253 1.841 ± 0.167 2.085 ± 0.089 2.308 ± 0.074

82.61 ± 2.911 47.75 ± 3.432 1.343 ± 0.037 0.456 ± 0.056 274.2 ± 9.37 2.415 ± 0.260 1.067 ± 0.096 0.746 ± 0.445 0.451 ± 0.018 40.51 ± 2.710 64.71 ± 3.046 37.54 ± 5.484 1.856 ± 0.165 1.961 ± 0.098 1.697 ± 0.098 *

76.93 ± 2.294 40.84 ± 2.177 1.487 ± 0.036 0.445 ± 0.081 264.3 ± 10.39 2.401 ± 0.381 1.169 ± 0.185 0.748 ± 0.082 0.463 ± 0.045 50.39 ± 2.278 * 58.09 ± 2.906 30.82 ± 1.054 1.903 ± 0.052 1.905 ± 0.057 2.390 ± 0.069 #

76.13 ± 2.327 40.49 ± 2.059 1.469 ± 0.057 0.452 ± 0.032 243.1 ± 9.76 2.128 ± 0.179 0.876 ± 0.074 0.720 ± 0.032 0.398 ± 0.017 39.36 ± 3.565 63.56 ± 2.484 39.19 ± 2.385 1.745 ± 0.129 1.944 ± 0.150 2.021 ± 0.071 #

* p < 0.05 compared to Control; # p < 0.05 compared to PAH.

Estimation of right ventricle systolic function was attainable by measuring TAPSE (tricuspid annular plane systolic excursion) values. TAPSE parameters of Control animals remained at normal range (TAPSEControl : 2.308 ± 0.074 mm), but significant decreases were found in values of PAH group animals (TAPSEPAH : 1.697 ± 0.098 mm) compared to the Controls. Values of WGLL-treated and

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sildenafil-treated animals were elevated in comparison to PAH group (TAPSEWGLL : 2.021 ± 0.071 mm, and TAPSESildenafil : 2.390 ± 0.069 mm, versus TAPSEPAH : 1.697 ± 0.098 mm). Diastolic function of the left ventricle was estimated using Doppler (PW) techniques, by determining E/A ratios at the mitral valve. E/A ratios were unaffected either by monocrotaline-injection, or by sildenafilor WGLL-treatment after 8 weeks. Int. J. Mol. Sci. 2017, 18, 1436

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2.3. Effects of Monocrotaline (MCT) and Treatments on Isolated Left Ventricular Function 2.3. Effects of Monocrotaline (MCT) and Treatments on Isolated Left Ventricular Function

Results of LV function obtained by isolated working heart method are shown in Figure 1. Results of LV function obtained by isolated working are shown Figure group 1. Monocrotaline treatment produced reduction in aortic flowheart (AF)method compared to the in Control Monocrotaline treatment produced reduction in aortic flow (AF) compared to the Control group (AFPAH : 27.38 ± 3.447 mL/min vs. AFControl : 55.33 ± 2.932 mL/min), and aortic flow of animals after (AFPAH: 27.38 ± 3.447 mL/min vs. AFControl: 55.33 ± 2.932 mL/min), and aortic flow of animals after WGLL treatment reached the Control values (AFWGLL : 54.36 ± 2.864 mL/min). Coronary flow (CF) WGLL treatment reached the Control values (AFWGLL: 54.36 ± 2.864 mL/min). Coronary flow (CF) and and Aortic tobe beunaffected unaffectedbybythe thetreatments treatments < 0.05). Heart Aorticpressure pressure(AoP) (AoP) were were observed observed to (p